This is an application for renewal of a grant to study to the mechanism and fidelity of nucleotide incorporation by the replicative polymerase of RNA viruses, the RNA-dependent RNA polymerase (RdRp). Over the past ten years, the world has witnessed the emergence of SARS, the spread of West Nile encephalitis, and the fear of a global flu pandemic. These diseases are caused by RNA viruses. In addition, the threat of intentional release of RNA viruses as weapons or agents of terror has increased substantially. The long-term goal of this research program is to develop strategies to treat and/or prevent RNA virus infection by targeting the RdRp. Our program has employed a prototypical RNA virus, poliovirus (PV), and its RdRp (3Dpol) as our model system. The previous funding period was devoted to the interrogation of the chemical mechanism for 3Dpol- catalyzed nucleotidyl transfer, elucidation of the structural basis for 3Dpol incorporation fidelity, and development of the tools to solve a crystal structure for 3Dpol in complex with primed template and nucleotide. We have made outstanding progress towards completion of all our aims. We have obtained new insight into the chemical mechanism for nucleotidyl transfer. We discovered a link between RdRp incorporation fidelity and pathogenesis. We discovered a connection between RdRp dynamics and incorporation fidelity. Together, our studies lead to the very provocative hypothesis that RdRp incorporation fidelity is a target for antiviral and vaccine development that will be elaborated during the next funding period. We will pursue the following specific aims: (1) Elucidate additional roles for the general acid in polymerase function; (2) Identify novel determinants and mechanisms of polymerase fidelity; and (3) Establish dynamics-function relationships for the RdRp.